The fundamental and essential process of translation is catalyzed by the ribosome, and this proposal focuses on biogenesis of the ribosome. Ribosomes are intricate RNPs, whose biogenesis involves the transcription, processing, modification and folding of rRNA and the dynamic binding of r-proteins and at least 40 assembly factors. Ribosomes have long been the benchmark for understanding functional capabilities of complex RNPs, and work to unravel the mechanisms of 30S subunit biogenesis will similarly increase our understanding of biogenesis of all cellular RNPs. Our long-term goal is to gain a detailed understanding of the factors and pathways involved in E. coli ribosomal subunit biogenesis. Since bacterial and eukaryotic ribosome biogenesis share several features, results from these studies will undoubtedly lead to new conserved features and new insights into biogenesis of eukaryotic ribosomes. Furthermore, since ribosome biogenesis is likely to be highly conserved among bacteria and is linked to pathogenicity of some bacteria, discoveries of bacterial- specific biogenesis events will yield a number of new possible antimicrobial targets and likely new insights into possible methodologies for medical manipulation to alter growth of pathogenic bacteria. Our approach involves the isolation of in vivo formed intermediates and subsequent analysis of the components, structures, and processing of these particles. Preliminary results have shown the existence of three distinct complexes, at least 30 potential assembly factors, three of which we have already confirmed as assembly factors. We will use these data to further interrogate ribosome biogenesis and to identify important steps in this process. We will use biochemistry, genetics, molecular biology and proteomics to study ribosomal subunit biogenesis intermediates and factors that are involved in these processes and these data will be used to develop a network of interactions important for this process in bacteria. These studies will not only result in novel targets for antibiotic screening and insights into bacterial drug resistance, but will also yield better understanding of the evolution of ribosome biogenesis, and the interplay of biogenesis with fundamental aspects of cell physiology.